Two component “mix and use” liquid thromboplastin reagent, methods of making, and methods of use thereof

ABSTRACT

What is described is a kit for preparing a liquid thromboplastin reagent for a prothrombin time assay. The kit simplifies and minimizes reagent preparation time and is stable for 2-5 years.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 15/429,480, which was filed on Feb. 10,2017. U.S. patent application Ser. No. 15/429,480 is incorporated hereinby reference. This application also claims the benefit of U.S.Provisional Application No. 62/294,367, filed Feb. 12, 2016, the entiredisclosure of which is herein incorporated by reference.

FIELD OF THE INVENTION

The field of the invention is related to reagents for preparing an assayfor assessing coagulation in a patient, more specifically reagents forprothrombin time assays, kits for coagulation assays, and methods ofpreparing such assays.

BACKGROUND OF THE INVENTION

The prothrombin time (PT) is used with patient plasmas to determineeither naturally occurring deficiencies in the extrinsic pathway ofblood coagulation, or those induced by the prescription of oral vitaminK antagonists (VKAs) such as coumadins. The PT is routinely performed inthe clinical setting using a manual or automated test method that mixesa working thromboplastin solution with human patient plasma collected ina stabilizing amount of chelator (e.g., citrate). Working thromboplastinsolutions are comprised minimally of tissue factor (natural (TF) orrecombinant (rTF)) in complex with a pro-coagulant lipid membranesurface, and calcium ions as a requisite cofactor to replace those thatwere chelated in the plasma when blood was drawn from a patient toprevent clotting. Working thromboplastin solutions are presented ineither liquid or freeze dried form.

Currently available liquid thromboplastins include all of the componentsin a single vial; this is disadvantageous because of limitations in longterm stability and shelf life which is no greater than 1 year whenmaintained at 2-8° C. Additionally, currently available freeze-driedthromboplastins are disadvantageous because they require significantpreparation time for reconstitution before use and introduce additionalvariability in precision of the PT measurement through thereconstitution process.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to a kit for preparing a liquidthromboplastin reagent for measuring prothrombin time in a patient. Inone embodiment, the kit includes a first container and a secondcontainer. The first container contains a coagulation factor III (tissuefactor, CD142) combined with a lipid mixture and a chelator. Thechelators may be, for example, but not limited to citrate, EDTA, EGTA,nitrilotriacetic acid (NTA), ethylene diamine,N-(2-Acetamido)iminodiacetic acid (ADA), tartrate, glycinate, BAPTA,oxalate, phosphate, diphosphate, polyphosphate, organic phosphonates,and combinations thereof. The concentration of the chelator is greaterthan about 0 mM to about 100 mM, greater than about 0 to about 50 mM,greater than about 0 mM to about 25 mM, about 10 mM to about 50 mM, forexample, and for EDTA, about 0.5 mM and for citrate, about 11 mM, forexample. The lipid mixture includes phospholipids, cholesterol, fattyacids, sphingolipids, mono-glycerides, di-glycerides, tri-glycerides,extracts of naturally occurring lipids and combinations thereof, forexample. The source of coagulation factor III (tissue factor) includesbut is not limited to a recombinant tissue factor or a native tissuefactor extracted from brain or other tissue.

The second container in this embodiment of the invention includes acalcium solution, typically in a buffer at a concentration ranging fromabout 5 mM to about 500 mM, about 5 mM to about 100 mM, about 10 mM toabout 75 mM, about 25 mM to about 50 mM, about 5 mM to about 25 mM, andabout 9 mM to about 15.5 mM, for example.

The kit according to this embodiment of the invention has a shelf-lifeof at least 2 to 5 years.

In another embodiment of this aspect of the invention, the kit has allof the same features as the kit described above with the exception of achelator.

In another aspect, the invention is directed to a method for preparing aliquid thromboplastin reagent for measuring prothrombin time in apatient. In one embodiment of the method of the invention, the firststep of the method provides coagulation factor III (tissue factor) asdescribed above in the ranges described above, a lipid mixture from thesources described above, and a chelator from the group described abovein the concentration ranges described above. In a second step, themethod provides a second container containing a calcium solution in theconcentration ranges described above. When a healthcare medicallaboratory professional prepares the liquid thromboplastin reagent formeasuring prothrombin time in a patient, the contents of the first andsecond containers are mixed together and may be used immediately.

The liquid thromboplastin reagent formed according to the method of theinvention has a shelf-life of at least 2 years.

In another embodiment of the method of the invention, the firstcontainer is the same as described above without a chelator.

In another aspect, the invention is directed to a method for prolongingthe shelf-life of a two-part thromboplastin reagent. The method followsthe steps provided above for preparing a liquid thromboplastin reagentfor measuring prothrombin time in a patient.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C show in tables 1-3 that the two component mix and use liquidthromboplastin kit according to the invention is stable for at least aminimum of 24 or more months;

FIGS. 2A and B illustrate chelator-induced protection againstprecipitation (i.e., instability) in the chelator-treated TF-lipidsamples according to the invention;

FIG. 3 includes Table 4 comparing chelator-induced stabilization ofchelator-treated TF-lipid samples as compared to the untreated TF-lipidcontrols;

FIG. 4 illustrates untreated (cloudy) samples in the bottom of the lefthand vials and chelator-treated (clear) samples in the bottom of theright hand vials; the data disclosed in FIG. 3 corresponds to the lefthand (untreated) and right hand (chelator-treated) vials.

FIG. 5A illustrates (PLase)-induced lipid degradation ofphosphatidylcholine under accelerating conditions (37° C. for 10 days)with or without EDTA chelator. FIG. 5B illustrates the percent change inPT in normal control plasma due to phosphatidylcholine degradation inReadiPlasTin® with and without chelator. Without chelator treatment,degraded phosphatidylcholine is exhibited as free choline above baselineat the highest PLase concentrations; while with chelator treatment,levels of choline detected are at baseline over all concentrations ofspiked PLase/calcium in the TF-lipid.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect the invention described herein is directed to extendingthe shelf-life of liquid thromboplastin, a reagent necessary for use inthe prothrombin time (PT) assay to test the capacity of a patient'sblood to coagulate. In the first embodiment of the invention, a calciumcomponent (e.g., calcium ions in a liquid component with or withoutbuffer) is separate from the tissue factor (TF)-lipid component (e.g.,TF-lipid in a liquid component with or without buffer) in a kit formeasuring prothrombin time. In this embodiment of the invention, thecalcium component is combined with the tissue factor-lipid componentjust prior to use and when mixed, a working thromboplastin is formedimmediately. Thus, according to the invention described herein, thecalcium component and the TF-lipid component of the thromboplastin areindependent reagents that do not come in contact with one another withinthe kit until the two components of the kit are mixed together beforeuse. The calcium and TF-lipid components of thromboplastin comprise thetwo components of the first embodiment of the kit according to theinvention.

In this first embodiment of the invention, working liquid thromboplastinis prepared prior to its use in a prothrombin time assay by the usercombining the liquid calcium component of the kit with a liquid TF-lipidcomponent of the kit, for example, by mixing 19 volume parts liquidcalcium with 1 volume part liquid TF-lipid. Working thromboplastinprepared by mixing the separate calcium and TF-lipid componentscomprises the first embodiment of “two-part mix (i.e., mixing of theliquid calcium component with the liquid TF-lipid component) and use”(i.e., immediate use of working thromboplastin to measure the PT)configuration of the invention.

The “mix and use” configuration of thromboplastin, as prepared above, isadvantageous over prior art liquid thromboplastin because it at leastreduces if not prevents divalent metal ion-dependent mechanisms of lipiddeterioration, such as divalent metal ion-dependent vesicle fusionleading to precipitation or divalent metal ion-dependent lipiddegradation (such as from contaminating lipid-degrading enzymes) whichcan reduce the performance or stability of the TF-lipid component of thekit. More importantly, because the “mix and use” configuration of thedisclosed liquid thromboplastin at least reduces if not prevents thedeleterious effects of TF-lipid storage in the presence of divalentmetal ions, the two component “mix and use” liquid thromboplastindescribed herein affords significantly increased shelf life over singlecomponent versions presently available, for example, at least up to twoyears of stability when maintained at 2-8° C.

The two component liquid thromboplastin invention described hereinmitigates the effects of stress on the TF-lipid reagent which are causedby but not limited to: thermal stress (e.g., such as improper storagetemperature), chemical stress, (e.g., trace contaminants such asphospholipases in the raw material stream used to make the TF-lipidreagent) or mechanical stress (e.g., such as that caused by large scalefiltration). These stresses are evident in the kit as precipitation inthe TF-lipid component, precipitation in the combined workingthromboplastin prepared from the mixture of the TF-lipid and calciumcomponents, or as large changes in the PT of control plasmas versusbaseline in the working thromboplastin prepared from the mixture of theTF-lipid and calcium components. In this two component embodiment, theliquid calcium component and the liquid TF-lipid component areindependently prepared as separate liquid thromboplastin components,each held in a separate compartment, e.g., separate containers, asdiscussed below. In separate compartments, the liquid calcium componentand liquid TF-lipid components of the embodiment comprise thethromboplastin-to-be prior to assembly by the user to form a workingliquid thromboplastin reagent.

In this embodiment of the invention, the separate liquid calcium andTF-lipid components have been engineered to be combined at 19 volumeparts calcium component to 1 volume part TF-lipid component so as toachieve a working thromboplastin reagent with the necessarycharacteristics for suitability in the PT assay. Among the necessarycharacteristics of the working thromboplastin are component compositionswhich produce PT results with normal citrated human plasma (i.e.,without extrinsic factor deficiencies or VKA or other anticoagulanttreatments) in the typical range of about 10-14 seconds (from initiationof coagulation to clot formation). In order to achieve PT results withnormal citrated human plasma in this range, working thromboplastinsrequire a calcium concentration sufficient to overwhelm the chelatorsstabilizing the citrated human patient plasma, typically 0-25 mM andTF-lipid concentrations typically in the range 0-1000 g/L.

In addition, embodiments of this invention described herein (such as 19volume parts calcium:1 volume part TF-lipid) are also contemplated so asto produce a working thromboplastin with the necessary characteristicsthat are the same as those described above. An alternate embodiment, forexample, includes a kit engineered with 1 volume part of calciumcomponent mixed with 19 volume parts of TF-lipid to make the workingthromboplastin. To produce a working thromboplastin with necessarycharacteristics in the PT assay, such a kit (1 volume part calcium:19volume parts TF-lipid) would require a much higher calcium concentrationin the liquid calcium component and much lower TF-lipid concentration inthe TF-lipid component versus the embodiment having 19 volume partscalcium:1 volume part TF-lipid. Both embodiments are contemplatedaccording to the invention since the embodiment employs separation ofthe calcium and TF-lipid components in the kit and since the workingthromboplastin performs equivalently in the PT assay with normalcitrated patient plasma.

In yet another (second) embodiment of the invention, one or morechelators are added to the liquid TF-lipid component in millimolarconcentrations (e.g., chelators greater than 0 mM to about 100 mM). Theone or more chelators protect the liquid TF-lipid component ofthromboplastin from divalent metal ion-dependent deterioration.Inclusion of the chelator or a chelating agent in the liquid TF-lipidcomponent of the kit's two components further extends the shelf-lifestability of the liquid TF-lipid component by providing protectionagainst divalent metal ion-dependent instability in the TF-lipidcomponent. The extended shelf-life stability of the chelator-treatedTF-lipid component further extends the shelf-life stability of the twocomponent “mix and use” liquid thromboplastin beyond two years, forexample between 2-5 years or longer, by providing protection againstdivalent metal ion-dependent instability, induced for example bychemical, mechanical or thermal stress to the TF-lipid component asdiscussed above.

In this second embodiment, the invention described herein is directed toextending the shelf-life of liquid thromboplastin by separating thecalcium component (e.g., calcium ions in a buffered liquid component)from the TF-lipid component in the kit and including a chelator orchelating agent in the separated liquid TF-lipid component to guardagainst divalent metal ion-dependent deterioration of the lipid. Suchdivalent metal ion(s) might be present in the TF-lipid component byhaving been introduced during the manufacturing process as traces in theraw material stream, airborne contaminants such as gypsum, or leachingfrom apparatus, vessels, or containers in contact with the TF-lipidduring manufacture.

A kit according to this second embodiment comprises a first containercomprising a tissue factor-lipid component in a buffer with one or morechelators at a concentration ranging between greater than 0 mM to about100 mM, and a second container comprising a calcium component (e.g. Ca++ions) in a buffer, for example, a calcium component, at a concentrationranging between about 5 mM to about 500 mM.

In the first embodiment described above where TF-lipid and calciumcomponents are separated but chelator is not present in the TF-lipid,the kit has a shelf-life of at least 1 to 2 years. For example, Tables1-3 in FIG. 1A-IC show the real time stability data for three distinctlots (FIG. 1A=Lot 1; FIG. 1B=Lot 2 and FIG. 1C=Lot 3) of the twocomponent “mix and use” liquid thromboplastin kit according to theinvention when the two component “mix and use” liquid thromboplastin kitwas stored at 2-8° C. for a minimum of 24 or more months.

Briefly, to generate the data in FIGS. 1A-C, the two component “mix anduse” liquid thromboplastin kits of the first embodiment (withoutchelators) described above were stored at 2-8° C., removed periodically(e.g., at various monthly time points up to 55 months), and tested forfunctional performance (Prothrombin Time (PT)(sec)) and fibrinogen(dFib) (mg/dL))) on an ACL TOP® instrument (Instrumentation Laboratory®)in normal control plasma (NC), low abnormal control plasma (Low Ab Con),and high abnormal control plasma (High Abn). Abnormal plasmas used inthese studies cause the PT to be prolonged greater than the 10-14seconds typical for normal control plasmas. The mean values at thesetimes were compared to those values acquired at baseline (0 months).Percent differences at various time points were calculated as follows: %difference:=(Stressed mean−Baseline mean)/(Baseline mean)×100. Valuesmeeting the specifications in Tables 1 to 3 in FIG. 1 (% difference <10%for the PT of NC; % difference <15% for the PT of Low Ab Con or HighAbn; % difference <15% for the dFib of NC, Low Ab Con or High Abn)indicate that the two component “mix and use” liquid thromboplastin kitis stable. Thus, the results summarized from Tables 1 to 3 in FIG. 1demonstrate that the embodiment described above of the two component“mix and use” liquid thromboplastin kit without chelators is stable forat least a minimum of 24 or more months.

In the second embodiment of the invention in which one or more chelatorsare added to the two component “mix and use” liquid thromboplastin kit(specifically, a chelator such as EDTA added to the liquid TF-lipidcomponent), the two component “mix and use” liquid thromboplastin kit isstable between 24 (2 years) and 60 months (5 years).

In this second embodiment of the invention, a kit is provided forpreparing a liquid thromboplastin reagent for measuring prothrombin timediscussed above, the kit comprises a first container comprising tissuefactor (TF)-lipid component and a chelator in a buffer. The chelatorconcentration is greater than about 0 mM to about 100 mM, greater thanabout 0 to about 50 mM, greater than about 0 mM to about 25 mM, about 10mM to about 50 mM, for example, and for EDTA, about 0.5 mM and forcitrate, about 11 mM, for example. The kit according to this embodimentof the invention further comprises a second container comprising acalcium component (e.g. Ca++ ions), such as, a buffered calciumcomponent, at a concentration ranging between about 5 mM to about 500mM.

In this second embodiment, the shelf-life of the kit including thecontents of the first container and the contents of the second containeris between about 2 to 5 years.

Tissue factor for the first and second embodiments directed to atwo-part liquid thromboplastin reagent according to the inventiondescribed herein can be selected from a recombinant tissue factor or anative tissue factor or a combination of both recombinant and nativetissue factor. Tissue factor is also known by other synonyms, such asbut not limited to, thromboplastin tissue factor, Factor III,coagulation factor III, CD142, or platelet tissue factor: synonyms forthe tissue factor are encompassed within the scope of this invention.

Typically, native tissue factor is extracted from brain or othertissues, such as such as brain, liver, kidney, heart, blood vessels,placenta or endothelial cells, platelets, blood cells (such as, but notlimited to, monocytes, neutrophils and other granulocytes), tumor cells,tissue factor-positive micro particles, endothelial cells orsub-endothelial cells (such as, but not limited to, smooth muscle cellsor fibroblasts), and egg, soy, plant tissues, yeast, and bacteria.

The concentration of calcium (e.g., a Ca++ ion) in solution for thefirst and second embodiments directed to a two-part liquidthromboplastin reagent according to the invention described hereinranges from about greater than 0 mM to about 500 mM. For example, thecalcium solution is greater than 0 mM to about 90 mM, greater than 0 mMto about 80 mM, greater than 0 mM to about 70 mM, greater than 0 mM toabout 60 mM, greater than 0 mM to about 50 mM, greater than 0 mM toabout 40 mM, greater than 0 mM to about 30 mM, greater than 0 mM toabout 20 mM, greater than 0 mM to about 10 mM, about 5 mM to about 100mM or about 10 mM to about 30 mM, about 10 mM to about 20 mM, or about 9mM to about 15.5 mM. The preferred ratio for calcium:TF-lipid is 19:1.

The chelator for the second chelator embodiment directed to a two-partliquid thromboplastin reagent according to the invention describedherein is selected from, but not limited to, citrate,ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid(EGTA), nitrilotriacetic acid (NTA), ethylene diamine, N-(2-Acetamido)iminodiacetic acid (ADA), diethylenetriaminepentaacetic acid (DTPA),tartrate, glycinate, BAPTA((1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), oxalate,phosphate, diphosphate, polyphosphate or organic phosphonates, orcombinations of the above.

The concentration of the chelator or the chelating agent (e.g., EDTA)for the second chelator embodiments directed to a two-part liquidthromboplastin reagent according to the invention described hereinranges from about greater than 0 mM to about 100 mM. For example, thechelating agent is in a concentration range of about 0 mM to about 100mM, greater than about 0 to about 50 mM, greater than about 0 mM toabout 25 mM, about 10 mM to about 50 mM, for example, and for EDTA,about 0.5 mM and for citrate, about 11 mM, for example.

The buffer in the tissue factor-lipid component for the first and secondembodiments directed to a two-part liquid thromboplastin reagentaccording to the invention described herein is selected from Tris or anybiological buffers or derivatives thereof, including but not limited to,Hepes, MES buffer, Bis-Tris buffer, citrate, ADA buffer, ACES buffer,PIPES buffer, imidazole/imidazolium buffer, Bis-Tris Propane buffer,maleic acid buffer, phosphate buffer, MOPSO buffer, BES buffer, MOPSbuffer, TES buffer, DIPSO buffer, MOBS buffer, TAPSO buffer, HEPPSObuffer, POPSO buffer, EPPS (HEPPS) buffer, Tricine buffer, Gly-Glybuffer, Bicine buffer, HEPBS buffer, TAPS buffer, AMPD buffer, TABSbuffer, AMPSO buffer, methylmalonate, diethylmalonate, or Glycine Amidehydrochloride buffer.

The calcium component buffer in the calcium component for the first andsecond embodiments directed to a two-part liquid thromboplastin reagentaccording to the invention described herein is selected from Tris or anybiological buffers or derivatives thereof, including but not limited to,Hepes, MES buffer, Bis-Tris buffer, citrate, ADA buffer, ACES buffer,PIPES buffer, imidazole/imidazolium buffer, Bis-Tris Propane buffer,maleic buffer, phosphate buffer, MOPSO buffer, BES buffer, MOPS buffer,TES buffer, DIPSO buffer, MOBS buffer, TAPSO buffer, HEPPSO buffer,POPSO buffer, EPPS (HEPPS) buffer, Tricine buffer, Gly-Gly buffer,Bicine buffer, HEPBS buffer, TAPS buffer, AMPD buffer, TABS buffer,AMPSO buffer, methylmalonate, diethylmalonate, or Glycine Amidehydrochloride buffer.

The lipid component of the tissue factor-lipid for the first and secondembodiments directed to a two-part liquid thromboplastin reagentaccording to the invention described herein affects functional activityand is made of, but not limited to, a mixture of phospholipids,cholesterol, individual fatty acids, sphingolipids, mono-, di-, ortriglycerides or lipid extracts of naturally occurring materialsincluding but not limited to egg, soy or other plant tissues, yeast,bacteria, or animal tissues such as heart, brain, liver, etc.

In another aspect, the invention is directed to a method for preparing aliquid thromboplastin reagent for measuring prothrombin time. The methodincludes providing a kit comprising a first container, the firstcontainer housing a liquid form reagent including tissue factor and alipid, e.g., a phospholipid, and providing a second container comprisinga calcium solution in a buffer (e.g., calcium-buffered component) at aconcentration ranging from about 5 mM to about 500 mM in the secondcontainer.

In a subsequent step, the liquid form reagent comprising tissue factorand lipids in the first container are mixed with the calcium solution inthe second container to form at room temperature the liquidthromboplastin reagent.

The shelf-life of the liquid form thromboplastin reagent without theaddition of a chelator is at least about 2 years.

In another aspect, the invention comprises a method for preparing aliquid thromboplastin reagent for measuring prothrombin time. The methodcomprises providing a kit comprising a first container. The firstcontainer comprises a liquid form reagent comprising a tissue factor, alipid, e.g., a phospholipid mixture, and a chelator (chelatorconcentration ranges from greater than 0 mM to about 100 mM) andproviding a second container comprising a calcium solution in a buffer(e.g., calcium buffered component) at a concentration ranging from about5 mM to about 500 mM.

In a subsequent step, the liquid form reagent comprising the tissuefactor, lipids, and a chelator is mixed with the calcium solution toform at room temperature the liquid thromboplastin reagent.

The shelf-life of the liquid form thromboplastin reagent including achelator described above is at least greater than 1 year and preferablybetween 2 to 5 years.

In another aspect, the invention is directed to a method for prolongingthe shelf-life of (or stabilizing) a two-part liquid thromboplastinreagent in a kit, comprising providing component (a) comprising acombination of tissue factor and phospholipid in liquid form, providinga chelator (b) and adding the chelator (b) to component (a), providing acomponent (c) comprising calcium in solution in a concentration range ofabout 5 mM to about 100 mM to form the prolonged shelf-life two-partliquid thromboplastin reagent having a chelator. The shelf-life of thetwo-part thromboplastin reagent having a chelator described herein is atleast greater than 1 year and at least about 2 years, preferably between2 and 5 years.

Advantages provided by the invention disclosed herein include but arenot limited to:

Providing kits and liquid reagents for a PT assay in which liquidreagents have a greater shelf-life than prior art liquid thromboplastinof at least more than one year and up to at least 5 years.

Providing kits and liquid reagents for a PT assay in which reagents areprotected against stress-induced (e.g., contaminant-induced mechanical,chemical, or thermal-induced stress) instability of reagents useful indetermining prothrombin time.

Simplifying and minimizing reagent preparation time for a PT assay.

EXAMPLES OF THE VARIOUS EMBODIMENTS OF THE INVENTION

The two component mix and use liquid thromboplastin according to theinvention described herein includes calcium ions (Ca++) in a componentseparate from the liquid tissue factor-lipid portion (native orrecombinant TF-lipid), and the working thromboplastin was prepared priorto use by dilution of the native or recombinant TF-lipid in a buffer(e.g., calcium buffered component). This “mix and use” configuration isunique in the marketplace for liquid thromboplastins, and keepsdeleterious lipid degrading mechanisms such as calcium-dependent vesiclefusion or metal ion-dependent lipid degradation from affecting thesusceptible lipids. In addition, to further protect against metal ionvesicle fusion leading to thromboplastin instability and precipitation,chelators such as EDTA and citrate may be introduced into the TF-lipidportion to inhibit this metal ion induced mechanism. The presence of themodest (i.e., millimolar) chelator levels in the reagent portion is madeirrelevant in the working thromboplastin because mixing with the excessof calcium in the component overrides the chelator levels in the finalworking thromboplastin). This protective mechanism using chelators isnot possible in prior art single component thromboplastin since chelatorconcentrations in a single component thromboplastin are required withinthe working thromboplastin to be in excess to overwhelm the citratechelator in human plasma, and therefore any and all chelators added tothe TF-lipid component would necessarily be overwhelmed by calciumlevels needed to activate coagulation. Moreover, the “mix and use”configuration of the liquid thromboplastin described herein minimizesreagent preparation time needed for to a ready-to-use thromboplastinreagent, where practically no incubation time is required before use ofthe thromboplastin reagent. This is a distinction from prior artthromboplastin reagents and is more user-friendly and efficient thanprior art thromboplastin reagents.

During the development of the two component mix and use liquidthromboplastin, some sources of instability in the TF-lipid portion wereidentified. Varying levels of trace phospholipase activities (e.g., ofphospholipases D, A1, A2 and C), phospholipase D activity in particular,were detected in the degraded TF-lipid component of aged kits. Thephospholipase D activity could be demonstrated as originating in the rTFraw material, and these were minimized in the raw material stream usinga pre-qualification assay for phospholipase D. Also, mechanical stress(such as from cartridge filtration) caused a temporary instabilityvisible during thermal stress (37° C.), which resolved itself withinfour months of storage at 2-8° C. This instability was accompanied by anincrease in the population of cloudy TF-lipid reagent vials with time at37° C. The effects of both these issues were mitigated by using mM(e.g., greater than 0 mM to about 100 mM) levels of chelator in theTF-lipid reagent vials. As illustrated in FIGS. 2A and 2B, inclusion ofa minimum of 0.5 mM EDTA (FIG. 2A) or 10.88 mM sodium citrate (FIG. 2B)completely eliminated the instability induced in ReadiPlasTin®, a PTreagent, (Instrumentation Laboratory Company, Bedford, Mass.) by thelarge scale filtration process in a dose-dependent manner.

Furthermore, referring to Table 4 in FIG. 3, the chelator presence alsoprovided a significant level of functional protection againstprolongation of the PT. Table 4 compares material filtered through a10-inch 0.2 micron spiral cartridge filter during production of theTF-lipid component for a manufactured lot “P4” (Lot N1042691: 19 partscalcium, 1 part TF-lipid, same concentrations (typically 9-15.5 mMcalcium in the diluent, with TF-lipid containing 3 g/L lipid and 3.375to 4.5 mg/L TF) which was treated with EDTA (upper table in FIG. 3) anduntreated with EDTA (lower table in FIG. 3) prior to stress at 37° C.for 10 days. After dilution with the matching calcium component (1 partTF-lipid, 19 parts calcium component) to make working thromboplastin,the EDTA containing samples showed significant protection againstfiltration induced susceptibility to stress, and gave prothrombin timesfor control materials as expected for unstressed two component mix anduse liquid thromboplastin. Thus, FIGS. 3 and 4 exemplify that a chelator(e.g., EDTA) stabilized the chelator-treated samples (e.g., the TF-lipidportion) and produced a robust stability as compared to the untreatedsamples. Most samples without chelator demonstrated precipitation andfailed the test. FIG. 4 illustrates the cloudy (precipitation) samplesin the bottom of the left hand vials which were untreated with chelator(left hand vials), and the clear samples (no precipitation) in thebottom of the right hand vials treated with a chelator.

FIG. 5 illustrates the protection chelators afford the TF-lipid portionof ReadiPlasTin from contaminating phospholipase (PLase) introduced intofreshly prepared reagent (TF-lipid lot N1065947). Fractions containingphospholipase D activity, detected as free choline from degradation ofphosphatidylcholine (Thermo Fisher, Amplex Red kit #A12219), wereisolated from Sf9 insect cell extracts, which is the same sourcematerial for the TF used in making ReadiPlasTin. The PLase-containingfractions were identified and isolated in the Superdex 200 gelfiltration elution profile (fractions 14-16, GE Healthcare Bio-Sciences,Pittsburgh, Pa. 15264-3065) distinct from TF fractions (fractions 6-12),combined and concentrated in a stirred cell concentrator over a YM-10(EMD Millipore Headquarters Billerica, Mass. 01821) diafiltrationmembrane by approximately 200-fold (˜0.5 mg/mL), stabilized to 5 mMcalcium using a 5-fold concentrated (“5×”) Amplex Red choline assaybuffer at 25 mM calcium (4 volumes concentrate+1 volume “5×”, thendiluted eight times at 1:10 each using “5×” Amplex Red choline assaybuffer diluted to 5 mM calcium). The resulting series of 10-fold dilutedstocks with PLase D activity were added to 1% of volume (10 μL into 1.0mL TF-lipid component) to adulterate the TF-lipid portion ofReadiPlasTin N1065947 vials which were pre-treated with or without 1.0mM EDTA chelator. After acceleration of the test by stressing 10 days at37° C., each of the vials was assayed for choline using a sensitiveAmplex Red PLase-D fluorescence assay (kit #A12219; Thermo FisherScientific Headquarters, MA 02451) to detect whether phosphatidylcholinein the TF-lipid component of the formulation had degraded to freecholine.

In the series where the TF-lipid component was untreated with chelator(FIG. 5A), choline was detected above baseline (i.e., at 1-18 μM) in thevials where the PLase was highest (5E-3 to 5E-5; i.e., 5×10-3 to 5×10-5)mg/mL, diamonds, FIG. 5A), but was undetectable (i.e., near 0 μM) invials treated with the chelator (squares, FIG. 5A). Similar protectionfor the TF-lipid component treated with chelator versus untreatedTF-lipid component was found for working thromboplastins prepared fromthese samples (FIG. 5B). Measurements of % difference from baseline forthe PT of Normal Control Plasma for the TF-lipid series treated withchelator (1 mM EDTA, squares, FIG. 5B) were low and constant across allconcentrations of PLase D contaminant (between −0% to −5%). Measurementsof the % difference from baseline for the PT of Normal Control Plasmafor the untreated (without EDTA) TF-lipid series (diamonds, FIG. 5) werehigher across the range of PLase D contaminant (−2% to −13%). And at thehighest levels exceeded specification for allowable PT % difference(<10% for Normal Control). The results illustrated in FIG. 5 provideevidence that use of chelator in the TF-lipid portion of ReadiPlasTinprotects against phosphatidylcholine phospholipid degradation caused byPLase contaminant under accelerating (e.g. thermally stressing)conditions.

While the present invention has been described in terms of certainexemplary embodiments, it will be readily understood and appreciated byone of ordinary skill in the art that it is not so limited, and thatmany additions, deletions, and modifications to the preferredembodiments may be made within the scope of the invention as hereinafterclaimed.

What is claimed is:
 1. A system comprising: a first compartmentcontaining a coagulation Factor III in a buffer combined with a lipidmixture and a chelator, wherein the chelator comprises at least one of:citrate, Ethylenediaminetetraacetic acid (EDTA), Ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) in aliquid form, Nitrilotriacetic acid (NTA), ethylene diamine,N-(2-Acetamido)iminodiacetic acid (ADA), diethylenetriaminepentaaceticacid (DTPA), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA), oxalate, triphosphate, diphosphate, polyphosphate, or organicphosphonates, and wherein the chelator has a concentration that iswithin a range of greater than 0 millimolar (mM) to 100 mM; and a secondcompartment containing a calcium component in a buffer at aconcentration ranging from about 5 mM to about 500 mM, wherein theconcentration of the calcium component in the second compartment is inexcess of the concentration of the chelator in the first compartment,and wherein the concentration of the calcium component is sufficient,upon mixing of contents of the first compartment with contents of thesecond compartment, to overwhelm the concentration of the chelator atleast to an extent that sufficient free calcium is available to activatecoagulation in a blood sample.
 2. The system of claim 1, wherein thesystem has a shelf-life of at least 2 years.
 3. The system of claim 1,wherein the lipid mixture comprises at least one of a recombinant tissuefactor or a native tissue factor.
 4. The system of claim 3, wherein thenative tissue factor is an extraction from at least one of a brain, aliver, a kidney, a heart, blood vessels, a placenta or endothelialcells, platelets, blood cells, tumor cells, tissue factor-positive microparticles, endothelial cells or sub-endothelial cells and egg, soy,plant tissues, yeast, or bacteria.
 5. The system of claim 3, wherein thenative tissue factor comprises an extraction from animal tissue.
 6. Thesystem of claim 1, wherein the lipid mixture comprises an extractionfrom animal tissue.
 7. The system of claim 1, wherein the concentrationof the calcium component is in a range of 9 mM to 15.5 mM.
 8. The systemof claim 1, wherein the concentration of the chelator is in a range of0.5 mM to 11 mM.
 9. The system of claim 1, wherein the lipid mixturecomprises at least one of phospholipids, cholesterol, fatty acids,sphingolipids, mono-glycerides, di-glycerides, tri-glycerides, or lipidextracts of naturally occurring materials.
 10. The system of claim 9,wherein the lipid extracts of naturally occurring materials comprise atleast one of egg, soy, plant tissues, yeast, bacteria, or animaltissues.
 11. The system of claim 1, wherein the system comprises a kit.12. The system of claim 1, wherein the first compartment comprises afirst container and the second compartment comprises a second container.13. A system comprising: a first compartment containing a thromboplastintissue factor in a buffer combined with a lipid in a liquid form and achelator, wherein the chelator comprises at least one of: citrate,Ethylenediaminetetraacetic acid (EDTA), Ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) in a liquid form,Nitrilotriacetic acid (NTA), ethylene diamine,N-(2-Acetamido)iminodiacetic acid (ADA), diethylenetriaminepentaaceticacid (DTPA), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA), oxalate, triphosphate, diphosphate, polyphosphate, or organicphosphonates, and wherein the chelator has a concentration that iswithin a range of greater than 0 millimolar (mM) to 100 mM; and a secondcompartment containing a calcium component in a buffer at aconcentration ranging from about 5 mM to about 500 mM, wherein theconcentration of the calcium component in the second compartment is inexcess of the concentration of the chelator in the first compartment,and wherein the concentration of the calcium component is sufficient,upon mixing of contents of the first compartment with contents of thesecond compartment, to overwhelm the concentration of the chelator atleast to an extent that sufficient free calcium is available to activatecoagulation in a blood sample.
 14. The system of claim 13, wherein thelipid comprises at least one of phospholipids, cholesterol, fatty acids,sphingolipids, mono-glycerides, di-glycerides, tri-glycerides, or lipidextracts of naturally occurring materials.
 15. The system of claim 14,wherein the lipid comprises an extract of naturally occurring materialscomprising at least one of egg, soy, plant tissues, yeast, bacteria, oranimal tissues.
 16. The system of claim 13, wherein the thromboplastintissue factor is extracted from animal tissue.
 17. The system of claim13, wherein the system is a kit.
 18. The system of claim 13, wherein thefirst compartment comprises a first container and the second compartmentcomprises a second container.
 19. A method comprising: at roomtemperature, mixing content of a first compartment containing a liquidform reagent with content of a second compartment containing a calciumsolution to form a liquid thromboplastin reagent at room temperature;wherein the liquid form reagent comprises a thromboplastin tissue factorin a first buffer, a lipid mixture, and a chelator comprising at leastone of: citrate, Ethylenediaminetetraacetic acid (EDTA), Ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) in aliquid form, Nitrilotriacetic acid (NTA), ethylene diamine,N-(2-Acetamido)iminodiacetic acid (ADA), diethylenetriaminepentaaceticacid (DTPA), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA), oxalate; triphosphate, diphosphate, polyphosphate, or organicphosphonates, and wherein the chelator has a concentration that iswithin a range of about 0 millimolar (mM) to about 100 mM; and whereinthe content of the second compartment comprises a calcium component in asecond buffer at a concentration range of about 5 mM to about 500 mM,wherein a concentration of the calcium component is in excess of theconcentration of the chelator, and wherein the concentration of thecalcium component is sufficient, upon the mixing of the content of thefirst compartment with the content of the second compartment, tooverwhelm the concentration of the chelator at least to an extent thatsufficient free calcium is available to activate coagulation in a bloodsample.
 20. The method of claim 19, wherein the liquid thromboplastinreagent has a shelf-life of at least 2 years.
 21. The method of claim19, wherein the thromboplastin tissue factor comprises at least one of arecombinant tissue factor and phospholipids, cholesterol, fatty acids,sphingolipids, mono-glycerides, di-glycerides, tri-glycerides, or lipidextracts of naturally occurring materials.
 22. The method of claim 21,wherein the lipid extracts of naturally occurring materials comprise atleast one of egg, soy, plant tissues, yeast, bacteria, or animaltissues.
 23. The method of claim 19, wherein the thromboplastin tissuefactor is an extraction from animal tissue.
 24. The method of claim 19,wherein the first buffer or the second buffer comprises at least one ofa Hepes, a MES buffer, a Bis-Tris buffer, citrate, an ADA buffer, anACES buffer, a PIPES buffer, an imidazole/imidazolium buffer, a Bis-TrisPropane buffer, a maleic acid buffer, a phosphate buffer, a MOPSObuffer, a BES buffer, a MOPS buffer, a TES buffer, a DIPSO buffer, aMOBS buffer, a TAPSO buffer, a HEPPSO buffer, a POPSO buffer, an EPPS(HEPPS) buffer, a Tricine buffer, a Gly-Gly buffer, a Bicine buffer, aHEPBS buffer, a TAPS buffer, an AMPD buffer, a TABS buffer, an AMPSObuffer, methyl malonate, diethylmalonate, or a Glycine Amidehydrochloride buffer.
 25. The method of claim 19, wherein the firstcompartment comprises a first container and the second compartmentcomprises a second container.
 26. The method of claim 19, wherein thelipid mixture comprises at least one of phospholipids, cholesterol,fatty acids, sphingolipids, mono-glycerides, di-glycerides,tri-glycerides, or lipid extracts of naturally occurring materials. 27.The system of claim 4, wherein the lipid mixture comprises at least oneof phospholipids, cholesterol, fatty acids, sphingolipids,mono-glycerides, di-glycerides, tri-glycerides, or lipid extracts ofnaturally occurring materials.
 28. The system of claim 13, wherein thethromboplastin tissue factor comprises at least one of a recombinanttissue factor and phospholipids, cholesterol, fatty acids,sphingolipids, mono-glycerides, di-glycerides, tri-glycerides, or lipidextracts of naturally occurring materials.